1. Field of the Invention
This invention relates to the quantitative determination of ligands, such as antibodies, antigens or haptens, in liquid media, including body fluids such as serum, based on specific binding assay techniques. In particular, the invention is directed to the detection of antibodies, antigens or haptens based on immunoassay techniques involving the use of labeled reagents, such as radiolabeled or enzyme-labeled reagents. The present invention provides a specific binding assay method of the heterogeneous type (i.e., wherein a bound-species of the labeled reagent is physically separated from a free-species thereof) for the simultaneous determination of each of a plurality of different ligands in a single liquid test sample.
2. Description of the Prior Art
A living system is able to detect, recognize and respond to the presence of foreign material (antigen) such as protein, virus, bacteria, and so forth, within that system. This response takes, inter alia, the form of producing an antibody specific for the particular antigen. There then occurs a specific reaction between the antibody and the antigen to form a complex. An antibody once produced is also capable of binding a hapten, i.e., a relatively small and simple compound which may be the determinant group of a given antigen, which hapten is capable of binding with the specific antibody but incapable itself of giving rise to the production of an antibody, unless it is bound to an antigenic carrier.
The binding interaction between an antigen or a hapten and its antibody is specific and sensitive. Other types of materials that participate in similar specific and sensitive binding interactions are enzymes and their substrates; materials such as hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances; and other substances known in the science. These specific and sensitive binding reactions have given rise to a rapidly emerging analytical technique known as the specific binding assay technique. Where a radioactive label is used and the binding reaction involved is immunological, the method is known as a radioimmunoassay (RIA) method. Recently, several alternative labeling materials have been reported for replacement of radioisotopes, including enzymes, coenzymes, enzyme substrates, enzyme modulators such as inhibitors and allosteric effectors, fluorescent molecules, luminescent molecules, and others.
In conventional specific binding assay techniques, the test sample is combined with reagent means of various compositions that include a labeled binding agent having a monitorable label component and a binding component which participates with other constituents, if any, of the reagent means and with the ligand under determination to form a binding reaction system having two species or forms of the labeled binding agent, a bound-species and a free-species. The relative amount or proportion of the labeled binding agent that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand to be detected in the sample.
As an illustration, a conventional competitive binding assay technique will now be described. In such a technique, the reagent means would comprise (1) a labeled binding agent in the form of the ligand to be detected (e.g., an antigen or hapten), such ligand constituting the binding component of the labeled agent, chemically linked to the label component (e.g., a radioactive atom or an enzyme) and (2) a solid-phase binding agent for the ligand (e.g., a solid-phase form of an antibody). Upon combination of the test sample and the reagent means, the ligand to be detected and the binding component of the labeled binding agent (in this illustration, a labeled form of the ligand) would compete in a substantially nondiscriminating manner for noncovalent binding to the solid-phase binding partner (in this illustration, an antibody). As a result, either the amount of labeled binding agent that would become bound to the binding partner (i.e., that results in the solid-phase bound-species) or that amount which would remain free (i.e., unbound to the binding partner and thus that results in the free-species) can be measured as a function of the amount of competing ligand present. The amount of labeled binding agent resulting in either species is determined by separating the solid-phase bound-species from the liquid-phase free-species and measuring, i.e., monitoring, the label component in one of the separated species.
As a further illustration, an alternative specific binding assay technique known as the "indirect solid-phase technique" will now be described. This type of assay is conventionally used where the ligand to be determined is a multi-valent entity such as antibodies. In the indirect solid-phase technique, the reagent means comprises (1) a labeled binding agent in the form of a binding partner (e.g., an antibody to an antibody) for the ligand to be detected (e.g., antibody) chemically linked to the label component, and (2) a solid-phase binding agent (e.g., an antigen corresponding to the antibody under assay bound to a solid-phase structure). The test sample is first incubated with the solid-phase binding agent whereby ligand from the sample becomes bound to the solid-phase binding agent, thus forming solid-phase ligand-binding agent complexes. Such complexes are then incubated with the labeled binding agent to form labeled solid-phase complexes (the bound-species) which are physically separated from the remaining liquid-phase (free-species) labeled binding agent. The amount of label associated with the solid-phase (bound-species) is a direct function of the amount of ligand in the test sample.
Attempts have been made to develop combined heterogeneous specific binding assays wherein a multiplicity of ligands are determined simultaneously in a single test sample. Such combined assay would result in a savings in time and cost over the performance of individual assays and would require a lesser volume of sample for assaying, which is an important consideration where the sample is a body fluid such as serum. Combined assays are of particular advantage where the assays are of a screening nature. An example would be the diagnosis of immunity of women to viruses and other antigens which are responsible for congenital malformations, such as Rubella, Cytomegalovirus, Herpes Simplex virus and the parasite toxoplasma. In a combined assay, the patient's immunity against two or more of these antigens, indicated by the presence of antibodies to such antigens in the serum of the patient, would be determined in a single test using a single serum sample. Such combined tests would be applied to wide-range immunological screening of women, e.g., at the earliest possible time during pregnancy or at a late stage of pregnancy as well as testing of both the mother and child after delivery. Such immunodiagnostic tests have hitherto been performed separately in respect of each of the above-mentioned antigens, each test requiring a separate sample of serum.
Several immunodiagnostic tests have been described for the separate detection and serological determination of Rubella virus specific antibodies and Cytomegalovirus (hereinafter "CMV") specific antibodies, including radioimmunoassay (RIA) and enzyme-immunoassay (EIA) tests. Voller and Bidwell [Br. J. Exp. Path. 56:338(1975) and 57:243(1976)] used enzyme-linked immunosorbent assay procedures for the separate determination of antibodies to Rubella and to CMV. In accordance with this procedure, polystyrene microplates coated with Rubella antigen were incubated with human serum containing Rubella antibody, followed by incubation with anti-(human globulin) labeled with alkaline phosphatase. The activity of the bound enzyme was measured colorimetrically.
U.S. Pat. No. 4,016,043 describes the determination of Rubella antibodies by forming, on a microtiter plate coated with Rubella antigen, a so-called "sandwich" consisting of the following sequence of layers: (solid-viral antigen)-antibody-(viral antigen-enzyme).
Kirsti et al [J. Clin. Microbiol. 4:117(1976)] describe an indirect solid-phase RIA procedure for detecting Rubella virus specific antibodies (IgG and IgM) in human serum. Purified Rubella virus was adsorbed onto polystyrene spheres and the antibodies which were bound to the virus after incubation with the serum were detected by subsequent binding of .sup.125 I-labeled anti-(human IgM) or anti-(human IgG). Similarly, Forghani et al [J. Clin. Microbiol. 4:470(1976)] used fixed, virus-infected cells as a source of antigen for binding the antibodies in the tested sera, while .sup.125 I-goat anti-(human IgG) was used for the detection of the specific antibodies (including Rubella antibody) which attached to the antigen. Similar indirect solid-phase RIA tests for CMV antibodies have recently been described by Forghani et al [Infect. and Immunity 14: 1184(1976)] and Knez et al [J. Immunol. 117:2006(1976)], the first using CMV-infected cells as the source of the antigen, and the other, viral soluble antigen fixed to microtiter plates. In both cases .sup.125 I-anti-(human globulin) was used for the detection of the antigen-antibody complexes formed.
In all of the above-described assays, a separate test specimen and separate assay procedure is necessary for determining each antibody. To the best of the applicants' knowledge, a combined specific binding assay for the simultaneous determination or more than one antibody species in a single sample was not known prior to their invention. [An assay similar to the present invention is reported in J. Immunol. Methods 26:381(1979) published after the filing date of applicants' priority Israel application.] However, certain attempts have been reported for developing combined heterogeneous specific binding assays in general.
U.S. Pat. Nos. 3,720,760 and 3,952,091 describe simultaneous multiple radioimmunoassays providing a qualitative indication of the presence of one or more of a specific group of antigens. The assays are merely simple "yes/no" screening tests which cannot distinguish one antigen from another and cannot provide quantitative results. If the test is positive, separate assays must then be performed in order to determine which particular antigen or antigens are in fact present (cf. col. 4, lines 56-64 in U.S. Pat. No. 3,720,760 and col. 1, lines 49-53 in U.S. Pat. No. 3,952,091).
Combined specific binding assays for related haptens using a different label for each hapten under assay are reported in U.S. Pat. No. 3,928,553 and Acta Endocrinol. 81:487-494(1976) for the thyroid hormones T-3 and T-4 and in German OLS No. 2,803,154 for vitamin B-12 and folate. A different type of label, i.e., .sup.125 I and .sup.131 I, is used for each of the haptens under assay. Such assays accordingly have the decided disadvantage of requiring separate or more involved instrumentation for measuring the several different labels necessary. A significantly more desirable combined assay would utilize the same label for all of the ligands under determination. Habermann et al in J. Clin. Chem. Clin. Biochem. 14:494-601(1976) describe a combined RIA procedure for T-3 and T-4 using the same label (.sup.125 I), however, a very complicated sequence of separation steps is required to isolate by column chromatography the labeled T-3 from labeled T-4 at the conclusion of the assay. A significantly more desirable combined assay would require no more than the conventional separation steps to isolate each labeled species from the others.